Alanine Screening Mutagenesis Establishes the Critical Inactivating Damage of Irradiated E. coli Lactose Repressor.

International audienceThe function of the E. coli lactose operon requires the binding of lactose repressor to operator DNA. We have previously shown that γ rradiation destabilizes the repressor-operator complex because the repressor loses its DNA-binding ability. It was suggested that the observed oxidation of the four tyrosines (Y7, Y12, Y17, Y47) and the concomitant structural changes of the irradiated DNA-binding domains (headpieces) could be responsible for the inactivation. To pinpoint the tyrosine whose oxidation has the strongest effect, four headpieces containing the product of tyrosine oxidation, 3,4-dihydroxyphenylalanine (DOPA), were simulated by molecular dynamics. We have observed that replacing Y47 by DOPA triggers the largest change of structure and stability of the headpiece and have concluded that Y47 oxidation is the greatest contributor to the decrease of repressor binding to DNA. To experimentally verify this conclusion, we applied the alanine screening mutagenesis approach. Tetrameric mutated repressors bearing an alanine instead of each one of the tyrosines were prepared and their binding to operator DNA was checked. Their binding ability is quite similar to that of the wild-type repressor, except for the Y47A mutant whose binding is strongly reduced. Circular dichroism determinations revealed small reductions of the proportion of α helices and of the melting temperature for Y7A, Y12A and Y17A headpieces, but much larger ones were revealed for Y47A headpiece. These results established the critical role of Y47 oxidation in modifying the structure and stability of the headpiece, and in reduction of the binding ability of the whole lactose repressor

Radiation damage to DNA–protein specific complexes: estrogen response element–estrogen receptor complex

The exposure of a DNA–protein regulatory complex to ionising radiation induces damage to both partner biomolecules and thus can affect its functioning. Our study focuses on a complex formed by the estrogen response element (ERE) DNA and the recombinant human estrogen receptor alpha (ER), which mediates the signalling of female sex hormones, estrogens. The method of native polyacrylamide retardation gel electrophoresis is used to study the stability of the complex under irradiation by low LET radiation (60Co gamma rays) and the ability of the separately irradiated partners to form complexes. The relative probabilities of ERE DNA strand breakage and base damages as well as the probabilities of damages to the ER binding domain are calculated using the Monte Carlo method-based model RADACK

Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA

Representative 3D-structures of complexes with DNA-bending proteins among the three domains of life.

<p>The dimeric bacterial protein IHF (PDB 1IHF) and the monomeric <i>Euryarchaeal</i> protein MC1 contact the concave side of the DNA curvature. In <i>Eukaryota</i> and <i>Crenarchaea</i> subdomain the proteins SRY (HMG-box protein) (PDB 1J46), Sul7d (PDB 1AZP) and Cren7 (PDB 3KXT) contact the convex side of the DNA curvature.</p

Proposed 3D model for MC1 bound to DNA.

<p>(A) Superimposition of two positions for double-strand DNA (orange and red spheres) after docking on the MC1 static molecular shape (green-blue, cartoon or spheres) by interactive driving of the flexible ligand through electrostatic potential of the protein, in accordance with the experimental data. (B) Ten final models for the DNA-MC1 complex (silver cartoon and colored cartoon respectively) proposed after all-atom reconstruction. Average DNA axis curvature measured by the Curves program on these models is 109±6°. (C) Superimposition of 2 of the 8 models of DNA-MC1 complex resulting from HADDOCK. The DNA angle curvature seems to be correlated to the position of the flexible protein LP5 loop: 122° for model 1 (blue) and 81° for model 8 (pink).</p

DNA-binding surface of MC1.

<p>The DNA-binding surface of MC1 is represented in green on the solvent-accessible surface. Residues which combine significant CSP and positive charge, or CSP and internal dynamics, are labeled.</p

Relative orientation of the DNA extremity with respect to MC1.

<p>Sequence of the two oligonucleotides DNA1* (top) and DNA2* (bottom) including a paramagnetic probe; the color-coded <b>T</b> (red or green) represents dT-EDTA-Mn²⁺. The effect of each paramagnetic probe is shown on the three-dimensional surface of MC1 in red and green respectively.</p

DNA bending ability of the WT and mutant MC1 proteins evaluated by EMSA.

<p>EMSA experiment was performed as described for K_Dapp measurement: 0.1 nM of 5′-[³²P]-labeled 26 bp DNA were incubated with the WT or mutant MC1 protein (at 20 nM final concentration, excepting R25A and R25Q mutant versions for which 100 nM was used). At equilibrium, assays were analyzed by EMSA as described in the <i>Materials & Methods</i> section. After 3 hours of electrophoresis, the gel was dried and visualized by autoradiography. The relative electrophoretic mobility of the protein/DNA complex provides an evaluation of the bending ability of the MC1 version considered. With this short DNA duplex, an apparent greater mobility of the nucleoprotein complex is expected for a protein with a greater DNA bending.</p